A method for screening strains phenotypically similar to known strains
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A strain and phenotype technology, applied in the field of strain screening and evaluation, can solve the problems of no relevant reports, long evaluation process, omission of high-quality strains, etc., achieving broad application prospects, wide screening range, and short time-consuming effects.
Active Publication Date: 2021-10-15
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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However, the evaluation process is relatively long, and there is no comprehensive quantitative comparison method for the results of various indicators. It can only rely on experience to make choices, which is not only time-consuming and labor-intensive, but also misses some high-quality strains.
Raman spectroscopy technology is an efficient compound information identification technology, which can provide information on the molecular composition and structure of compounds. Raman spectrum collection of a cell will obtain molecular fingerprint information (Raman group) of all substances in the entire cell. The Raman group can reflect the phenotypic information of cells in a specific environment, and has attracted more and more attention. It is used to distinguish different stages of cells, identify cell types, and evaluate the effect of drugs. Whether the Mann group can be used as a detection method for strain selection with excellent performance has not yet been reported
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Embodiment 1
[0038] Example 1. Yeast cell phenotype evaluation based on Raman group similarity analysis (phenotype is known)
[0039] 1) Lager yeast cell culture
[0040]Four Lager yeast strains with known phenotypes (#21, #25, #32, #45) were used to evaluate the phenotypic similarity based on single-cell Raman group analysis, among which #21 was an inferior strain, and #25 was an Medium-sized strains, #32 and #45 are high-quality strains. After the four strains were activated in standard yeast medium, the EBC tube fermentation experiment was carried out. Take 1 mL of each post-ripening bacterial liquid, collect by centrifugation at 4500 rpm for 1 min, and resuspend with 1 mL of ddH2O Bacteria, after repeating 3 times, dilute the sample 100 times, draw 2uL with a pipette gun, spot on a CaF2 glass slide, and air-dry at room temperature. Among them, high-quality strains: the fermentation degree is greater than 65%, the maturity of acetaldehyde is less than 15ppm, the diacetyl is less than 6...
Embodiment 2
[0047] Example 2, yeast cell phenotype evaluation based on Raman group similarity analysis (phenotype unknown)
[0048] 1) Lager yeast cell culture
[0049] Randomly pick 8 clones from the plate to be screened as the unknown strains to be evaluated (#21_1~8), and select the high-quality strain #32 as the control strain at the same time. For the fermentation experiment, take 1 mL of each post-ripening bacterial liquid, centrifuge at 4500 rpm for 1 min to collect, resuspend the bacterial cells with 1 mL ddH2O, repeat 3 times, dilute the sample 100 times, draw 2uL with a pipette gun, and spot the sample on a CaF2 glass slide , air-dried at room temperature.
[0050] 2) Single-cell Raman group measurement
[0051] Place the air-dried CaF2 slide on the stage of a micro-Raman spectrometer, and randomly select single cells under the field of view of a 100-fold objective lens, and collect Raman spectrum signals. A 100mW 532nm laser is selected, and the filter is set to Set the OD t...
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Abstract
A method for screening strains with similar phenotypes to known strains, comprising culturing the strains to be selected and the known strains to the same growth period; removing the culture medium after sampling respectively, washing the strains with a low Raman background solution and resuspending; The to-be-selected strains and the known strains are respectively spotted on the sample sheet with a low Raman background; single cells are randomly selected from the to-be-selected strains and the known strains respectively, and the Raman group signals are collected, and the number of single cells selected is not less than 10 Collect the background signal next to the single cell and subtract the background signal; perform baseline processing on the single-cell Raman group data after deducting the background signal of the candidate strain and the known strain respectively, and obtain the corresponding candidate strain and known strain Single-cell Raman group pretreatment results; statistical analysis algorithm is used to analyze the similarity of the single-cell Raman group pretreatment results, and the similarity between the candidate strain and the known strain of the single-cell Raman group is greater than 85%. strains with similar phenotypes to known strains.
Description
technical field [0001] The invention relates to the field of screening and evaluation of strains, specifically, a method for screening strains with phenotypes similar to known strains by comparing the similarity of single-cell Raman groups between candidate strains and known strains. Background technique [0002] Germplasm resources with excellent and stable properties are the soul and lifeblood of the fermentation industry. The quality of fermentation is closely related to the quality of strains. However, strains are easily affected by the external environment during long-term continuous cultivation and storage. Mutations cause the loss of excellent phenotypes, changes in some important properties such as fermentation power and suspension, and the aging and degradation of strains will seriously affect the quality and economic benefits of products. Therefore, in order to ensure the stability of fermentation quality, it is necessary to rejuvenate the strains regularly, and to...
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